Asbestos exposure, pleural mesothelioma, and serum osteopontin levels

ABSTRACT

The present invention provides diagnostic methods based on the serum levels of osteopontin.

BACKGROUND

1. Technical Field

The present disclosure relates to occurrence of osteopontin in tissuesand fluids of subjects afflicted with pleural mesothelioma, anddiscloses serum osteopontin levels in three populations:asbestos-exposed subjects without malignancy, unexposed subjects withoutmalignancy, and asbestos-exposed subjects with pleural mesothelioma.

2. Related Art

Pleural mesothelioma is an asbestos-related malignancy with a mediansurvival of 8-18 months (Martino D et al., Clin Lung Cancer 2004;5(5):290-298). Retrospective studies of small numbers of pleuralmesothelioma patients have attempted to define biomarkers that predatesymptoms in a population at high risk for pleural mesothelioma. Thesemarkers include tissue polypeptide antigen, carcinoembryonic antigen,hyaluronic acid and ferritin, hyaluronic acid alone, cytokeratinsincluding Cyfra 21™, CA-125, and mesothelin related protein (SMRP)(Ebert W, et al., Anticancer Res 1997; 17(4B):2875-2878; Pettersson T,et al. Chest 1988; 94(5):1037-1039; Frebourg T, et al. Cancer 1987;59(12):2104-2107; Roboz J, et al. J Natl Cancer Inst 1989;81(12):924-928; Chiu B, et al, Cancer 1984; 54(10):2195-2199; Boersma A,et al. Bull Physiopathol Respir (Nancy) 1980; 16(1):41-45; Hellstrom PE,et al. Scand J Respir Dis 1977; 58(2):97-102; Thylen A, et al. Cancer2001; 92(5):1224-1230; Pluygers E, et al. Cancer Prev 1992;1(2):129-138; Lee Y C, et al. Aust N Z J Med 1999; 29(6):765-769;Paganuzzi M, et al. Chest 2001; 119(4):1138-1142; Schouwink H, et al.Lung Cancer 1999; 25(1):25-32; Marukawa M, et al. Acta Med Okayama 1998;52(2):119-123; Bonfrer J M, et al. Anticancer Res 1997;17(4B):2971-2973; Almudevar BE, et al. Histopathology 1997;31(3):267-273; Robinson B W, et al. Lancet 2003; 362(9396): 1612-1616).

The lifetime risk for pleural mesothelioma in an asbestos-exposedpopulation is 4.5-10%. Workers at risk for high exposure include miners,factory workers, carpenters, electricians, ship fitters, shipelectricians, boilermakers, insulation manufacturers, railroad workers,gas mask manufacturers, and pipe insulators (Roggli V L, et al.Ultrastruct Pathol 2002; 26(2):55-65). It has been estimated that asmany as 7,500,000 construction workers in the United States have usedasbestos construction materials for fireproofing buildings, acousticcontrol, duct work and pipe and boiler instillation (Schottenfeld D, etal. Cancer Epidemiology and Prevention. Philadelphia: W.B. Saunders Co.,1982). Moreover, asbestos is still a hazard for an estimated 1.3 millionworkers in the construction industry in the United States and forworkers involved in maintenance of building and equipment (U.S.Department of Labor OSaHA. Safety and Health Topics: Asbestos. 2003).

At present, there are no economically feasible, validated modalities toscreen all these individuals potentially at risk when the estimatednumber of new cases of mesothelioma in the United States per year isonly 2500-3000 (age adjusted incidence of 2/100,000) (Weill H, et al.Occup Environ Med 2004; 61(5):438-441). However, the incidence ofmesothelioma is rising in and outside the United States, includingCanada and Australia. Median survival in pleural mesothelioma is 9-12months from diagnosis; in advanced cases, resection of the tumor canprolong survival for about three months. Patients with stage IA disease,however, can survive 5 or more years if the tumor is promptly resected.Unfortunately, the difficulty in detecting early disease means that lessthan 5% of patients with pleural mesothelioma present in stage IA; infact 85% of patients first present once they are exhibiting symptoms ofthe disease. Hence, a marker or series of biomarkers that can predictthe development of mesothelioma or detect pleural mesothelioma in itsearly stages in asbestos-exposed populations would be of considerablevalue.

BRIEF SUMMARY

The present disclosure relates to the use of osteopontin as anoninvasive marker for mesothelioma in human subjects. Osteopontinlevels are elevated in serum of patients with mesothelioma, andmeasurement of these levels can be carried out conveniently for thepurpose of diagnosis, prognosis and therapy.

In one of its aspects, the disclosure provides a method of diagnosingmesothelioma by detecting a level of osteopontin in a bodily fluid orother biological sample of a patient, such as blood, serum, sputum,bronchial or alveolar lavage, a lung aspirate, a pleural fluid sample,or another sample derived from or contacted with lung tissue, andcomparing the level with a predetermined value or values or with acomparable sample obtained from another patient of known mesotheliomastatus.

In yet another aspect, the disclosure provides a method for monitoringthe response of a mesothelioma to radiation or chemotherapy, comprisingdiagnosing the mesothelioma, and measuring osteopontin levels before,during and/or after the radiation or chemotherapy treatment.

In still another aspect, the disclosure provides a method of monitoringthe response of a patient to tumor therapy, comprising determining alevel of osteopontin in a bodily fluid obtained from the patient priorto therapy, and at various times during and after therapy, and comparingthe level to one another or to a predetermined value or values that areindicative of the presence of mesothelioma.

The disclosure also provides a kit containing at least one binding agentfor detecting osteopontin and calibration means for comparing the levelof osteopontin with a predetermined value or values corresponding topresence of mesothelioma. Examples of suitable calibration means includestandardized osteopontin in amounts corresponding to a predeterminedvalue or range of values, instructions for using the kit, and adescription of osteopontin levels in various bodily fluids correspondingto negative and positive results for mesothelioma.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are graphs that illustrate validation of the naturalhistory for the test set of 76 mesotheliomas. FIG. 1A shows the mediansurvivals for the Stage I (solid line), Stage II (long dashes), andStage III (short dashes) patients. FIG. 1B shows the median time toprogression for the Stage I (solid line), Stage II (long dashes), andStage III (short dashes) patients.

FIG. 2A is a photograph illustrating osteopontin immunohistochemistry inpleural mesotheliomas at magnification×100. FIG. 2B is a photographillustrating osteopontin immunohistochemistry in pleural mesotheliomasat magnification×400.

FIGS. 3A-3D are bar graphs that illustrate the influence of asbestosdemographics on serum osteopontin levels. FIG. 3A illustratesosteopontin levels in asbestos exposed subjects (E) and age-matchedcontrols (U) for subjects younger than 50, between the ages of 50 and60, or older than 60 years (y). FIG. 3B illustrates osteopontin levelsin asbestos exposed subjects and age-matched controls by gender: (F)female and (M) male. FIG. 3C illustrates the level of osteopontin withyears (y) of asbestos exposure. FIG. 3D illustrates the degree ofradiographic abnormality in relation to osteopontin levels.

FIGS. 4A-F are graphs illustrating the ROC (Receiver OperatingCharacteristics) curves for mesothelioma versus asbestos exposedpopulations. FIG. 4A illustrates all 76 mesotheliomas versus 69 asbestosexposed. FIG. 4B illustrates the high sensitivity for early detection ofmesothelioma at a cutoff level of 10.9 ng/ml. FIG. 4C illustrates thecut-off for minimization of total classification error. FIG. 4Dillustrates stage I mesotheliomas versus asbestos exposed subjects. FIG.4E illustrates high sensitivity for early detection of Stage Imesothelioma versus asbestos exposed subjects at cutoff level of 9.5ng/ml. FIG. 4F illustrates the cut-off for minimization of totalclassification error for Stage I versus asbestos exposed.

DETAILED DESCRIPTION

The present disclosure provides new evidence that osteopontin is auseful biomarker for mesothelioma, including pleural mesothelioma, andmore specifically provides comparison of serum levels of osteopontin ina cohort of patients with asbestos-related non-malignant disease(hereafter called the asbestos-exposed group) with preoperative levelsin surgically treated pleural mesothelioma patients. Populations withasbestos-related nonmalignant disease are an ideal cohort in which tostudy biomarkers for the early detection of pleural mesothelioma,because they are at a high risk for development of the tumor; they havehad a measurable, identifiable exposure to the carcinogen; reside indefined and well studied geographical regions; and comply in long-termfollow-up studies (Cullen M R et al., Am J Epidemiol 2005;161(3):260-270).

In prior work, we used gene expression arrays to predict survival andrecurrence patterns in pleural mesothelioma (Pass H I, et al. ClinCancer Res 2004; 10(3):849-859) and to seek markers that would be usefulfor screening and diagnosis of pleural mesothelioma. The most promisingbiomarker was osteopontin, a glycoprotein that is overexpressed in lung,breast, melanoma, colorectal cancer, gastric cancer, and ovarian cancer(Fedarko N S, et al. Clin Cancer Res 2001; 7(12):4060-4066; Singhal H,et al. Clin Cancer Res 1997; 3(4):605-611; Denhardt D. J Invest Dermatol2005; 124(5):xvi-xviii; Ding L, et al. Zhonghua Wai Ke Za Zhi 2002;40(10):773-775; Ue T, et al. Int J Cancer 1998; 79(2):127-132; SchorgeJO, et al. Clin Cancer Res 2004; 10(10):3474-3478; Brakora K A, et al.Gynecol Oncol 2004; 93(2):361-365; Kim J H, et al. JAMA 2002;287(13):1671-1679). Osteopontin mediates cell-matrix interactions andcell signaling through binding with integrin and CD44 receptors (Wai PY, et al. J Surg Res 2004; 121(2):228-241), and is regulated by proteinsin cell signaling pathways that are associated with asbestos-inducedcarcinogenesis. Moreover, high levels of osteopontin correlate withtumor invasion, progression, and metastases. Sandhu reported thatosteopontin is upregulated in asbestos-induced tumors in a rat model ofasbestos carcinogenesis and in cells treated with asbestos in vitro(Sandhu H, et al. Carcinogenesis 2000; 21(5):1023-1029).

Osteopontin refers to a secreted highly acidic glycophosphoproteincharacterized by a conserved GRGDS amino acid sequence that includes,without limitation, the proteins described in Denhardt, D. T and Guo, X.FASEB J. 7:1475-82 (1993); and Denhardt, D. T. et al (Eds),“Osteopontin: Role in Cell Signalling and Adhesion,” Ann. NY Acad Sci.(1995) and homologous proteins. As used herein, the term encompassesvariants and fragments of osteopontin, i.e., naturally occurring formsof osteopontin that are substantially similar but nonidentical todescribed osteopontin in sequence and/or length and are capable ofsubstituting for osteopontin in a specific binding interaction, asdescribed below.

Human osteopontin has been completely sequenced and both polyclonal andmonoclonal antibodies that recognize this protein are commerciallyavailable (e.g., from R&D Systems Inc., Minneapolis, Minn.; and AbcamLtd., Cambridge, Mass.). Methods for detecting this protein have beendescribed in the art and ELISA assays for quantitating the human proteinare commercially available (Assay Designs Inc., Ann Arbor, Mich.). It ispossible that the osteopontin in biological fluids and samples may bepartially degraded or in a modified form due to processing by the body.For the purposes herein, it will be understood that assays fordetermining osteopontin levels which detect fragments of osteopontin ormodified forms of osteopontin are included within the scope of thedisclosure.

The results of the present study are described in the Examples. Briefly,there were no statistically significant differences in serum osteopontinlevels between age-matched asbestos-exposed and unexposed subjects (30±3ng/ml vs 20+4 ng/ml, respectively, p=0.06). Within the asbestos-exposedgroup, elevated serum osteopontin levels were associated with pulmonaryplaques and fibrosis (56±13 ng/ml) but not with normal radiologicalfindings (21±5 ng/ml), plaques (23±3 ng/ml), or fibrosis (32±71 g/ml),p=0.004. Serum osteopontin in the group with pleural mesothelioma was133±10 ng/ml, and 30±3 ng/ml in the 69 asbestos-exposed subjects(p<0.001). Immunohistochemistry revealed osteopontin that was limited totumor cells in 36/38 pleural mesotheliomas. ROC analysis of serumosteopontin levels comparing the asbestos-exposed non-malignancy groupto the mesothelioma group had a sensitivity and specificity of 77.6% and87% respectively at a cut-off of 48.4 ng/ml. Subgroup analysis comparingStage I mesotheliomas to asbestos-exposed subjects revealed asensitivity and specificity of 84.6% and 88.4% at a cut-off of 62.4ng/ml. Serum osteopontin levels therefore can distinguish asbestosexposure without malignancy from asbestos exposure with pleuralmesothelioma.

Thus, we found that osteopontin levels in the 69 members of theasbestos-exposed group did not differ from age matched controls, andthat osteopontin levels within this group are a reflection ofoccupational exposure duration and of radiographic abnormalities. Incontrast, as compared with the asbestos-exposed group, the osteopontinlevels in serum from patients with pleural mesothelioma weresignificantly higher. Careful documentation of the type of exposurehistory was accomplished using a standardized occupational/environmentalquestionnaire, and B reader interpretation of the radiographs allowedclassification of the asbestos-exposed population by important riskfactors for pleural mesothelioma, including the duration of theexposure. A multiple regression analysis revealed that duration ofexposure and radiographic findings were the most important influences onserum osteopontin levels, and that longer exposure and radiographicabnormalities were significantly associated with elevated osteopontinlevels. Fibrotic changes but not pleural plaques were associated withelevated levels. The combination of radiographic findings with serumlevels of osteopontin allow a practitioner or diagnostician to stratifyasbestos-exposed populations with regard to risk for pleuralmesothelioma; close surveillance might be indicated in workers with along exposure history, pleural plaques and fibrosis, and an elevatedserum osteopontin level.

An important result of this study is the ability of ELISA assays forosteopontin to identify early-stage pleural mesothelioma (Stage I). Thisfinding has immediate clinical applications and industrial applicabilitybecause therapy is likely to influence survival of patients with Stage Ipleural mesothelioma. Furthermore, immunohistochemistry showed thatosteopontin is present in the tumor cells of pleural mesothelioma andnot in the stroma, which supports the specificity of osteopontin fortransformed mesothelial cells.

Osteopontin is being investigated as a biomarker in other types ofcancer, so methods of detection using this protein are available in theart. Using immunohistochemistry, Coppola (Coppola D, et al. Clin CancerRes 2004; 10(1 Pt 1): 184-190) described high staining levels ingastric, colorectal, pancreatic, lung, and ovarian carcinomas amongothers, and a strong correlation with pathologic stage. Schneider(Schneider S, et al. Clin Cancer Res 2004; 10(5):1588-1596) found thathigh tissue osteopontin levels, measured by a real time polymerase chainreaction, correlated with decreased survival in resected non small celllung cancer. In both pancreatic cancer and breast cancer, serumosteopontin levels measured by ELISA were elevated in patients with newor progressive neoplasm (Koopmann J, et al. Cancer Epidemiol BiomarkersPrev 2004; 13(3):487-491; Martinetti A, et al. Endocr Relat Cancer 2004;11(4):771-779).

Our data indicate that serum osteopontin levels could discriminatebetween asbestos-exposed persons and those with early stage pleuralmesothelioma, independent of the histology of the mesothelioma.Moreover, the AUC approaching 0.9 implies that osteopontin has apositive predictive power equivalent to that of CA 125 for ovariancancer (McIntosh M W, et al. Gynecol Oncol 2004; 95(1):9-15).Osteopontin levels, however, are also elevated in other types ofcancers, including gastrointestinal, laryngeal, and urinary neoplasms,and these malignancies have been associated weakly with asbestosexposure. The hypothesis that the osteopontin level may be increased inasbestos workers who develop malignancies other than mesothelioma isbeing investigated. Nevertheless, based on data disclosed herein,asbestos workers with high osteopontin levels who do not appear to havea mesothelioma should be investigated to rule out other malignancies,including lung malignancies, and so elevated osteopontin can serve as amarker for asbestos-related injury that could progress to lung cancer.

In addition to being a useful molecular marker for mesothelioma,osteopontin levels can be used prognostically to assess the likelihoodof relapse after therapy. Based on the data herein, the presentdisclosure provides a noninvasive method for detecting mesothelioma inpatients and for identifying patients at high risk for mesotheliomarecurrence.

Because osteopontin is secreted into bodily fluids, e.g., blood (plasma,serum), lymph, pleural effusion, urine, bile, milk, saliva, tears andothers, the level of osteopontin in a patient can be detected andmonitored using noninvasive procedures. A level of osteopontin in abodily fluid, preferably blood, may be detected by any protein chemistryanalytical techniques or bioassays capable of identifying andquantitating osteopontin. These methods are well-known in the art andare routinely used by those of ordinary skill in protein analysis and/orbioassay methodology. A preferred method involves the use of a bindingagent that reacts with osteopontin (or with a variant or fragmentthereof) in a highly selective manner. The binding agent itself maycontain a reporter group (e.g., a radioisotope, a fluorescent compound,a fluorescence emitting metal of the lanthanide series, achemiluminescent or phosphorescent molecule, a paramagnetic group, or anenzyme). Alternatively, a detecting reagent containing a suitablereporter molecule, and capable of binding the binding agent osteopontincomplex, may be used (e.g., an anti-immunoglobulin, protein A, proteinG). Examples of useful binding agents include antibodies, receptors,ligands or carrier molecules. Preferably, detection is carried out usingimmunoassay methods and immunoreagents that are well known in the art(Harlow and Lane, “Antibodies: A Laboratory Manual” Cold Spring HarborLaboratory), 1988. Further details of assays relevant to osteopontindetection are disclosed in, for example, U.S. Patent Publication20030044862.

The term antibody is intended to refer to intact antibody molecules andantigen-binding fragments such as Fab and F(ab′) that are produced byproteolytic cleavage of intact antibodies. Monoclonal antibodies orpolyclonal antibodies that are directed against one or more epitopes ofosteopontin can be used in the methods described herein. Polyclonalantibodies to osteopontin can be obtained from the sera of animals thatare immunized with osteopontin or from commercial sources. Monoclonalantibodies can be prepared by methods known to those skilled in the art(Kohler and Milstein, Nature 256:495-497, 1975).

Immunoassays are carried out in solution or, preferably, on a solidphase support that is capable of binding antigen or antibody. In atwo-antibody sandwich type immunoassay, purified antibody is bound to asolid support and the support is contacted with the test fluid samplefor sufficient time to allow the antigen in the sample (i.e.,osteopontin) to bind to the antibody. After washing to remove unboundproteins, a second antibody is allowed to bind to the antigen. Thisantibody is labeled with a reporter group and is directed against anepitope on the antigen that differs from and is nonoverlapping with theepitope bound by the immobilized antibody. After washing, the amount oflabeled second antibody bound to the solid support is measured. Eithermonoclonal antibodies or affinity-purified polyclonal antibodies can beused for this assay. The detection limit of the assay is typically about0.01-0.1 ng antigen. The sensitivity of the assay can be varied bychoice of a suitable label.

One suitable assay for use in the practice of this disclosure is atwo-antibody sandwich type assay in which a secondary antibody is linkedto an enzyme reporter group (ELISA). When exposed to its substrate, theenzyme will produce a product that can be detected by spectroscopicanalysis or by another quantitative analytical method (e.g., afluorescent, chemiluminescent, bioluminescent, phosphorescent orradiolabeled product). Suitable enzymes include, without limitation,alkaline phosphates, glucose oxidase, β-galactosidase, catalase, malatedehydrogenase, horseradish peroxidase, yeast alcohol dehydrogenase, andothers.

Alternatively, the detection and quantitation of osteopontin in thefluid may be carried out using an antigen capture assay in which asubsaturating amount of unlabeled antibody (polyclonal, high affinitymonoclonal or pooled monoclonal antibodies) is bound to the solidsupport, the sites for protein binding are blocked with a suitableblocking buffer (e.g., 3% BSA in PBS) and a fluid sample containing afixed amount of labeled purified antigen (selected to provide sufficientsignal within the linear range of binding to antibody) is added andallowed to bind to the antibody. After washing, the amount of labeledantigen is measured. The relative levels of antigen in different fluidsamples can be determined, e.g., by assaying serial dilutions of eachfluid sample and comparing the midpoints of the titration curves. Theabsolute amount of antigen in the sample can be determined by comparingthe measured values with values obtained using known amounts of pureunlabeled antigen in a standard curve. The binding reaction betweenantibody and fluid sample containing labeled and unlabeled osteopontinis conveniently carried out in a microtiter plate. Alternatively, thebinding may be carried out in solution and the complexes separated fromthe reaction mixture by contacting the reaction mixture with immobilizedanti-immunoglobulin antibodies or proteins that are specific for animmunoglobulin, e.g., protein A or protein G.

An alternative method for detecting the level of an antigen in a fluidsample is to bind the fluid sample directly to a solid support, removeunbound proteins by washing, add an antibody specific for that antigenand allow it to bind. After removing unbound antibody by washing, theamount of antibody bound to the solid support is determined using alabeled secondary immunoreagent, e.g., a labeled anti-immunoglobulinantibody, protein A or protein G. This method is not useful if theantigen makes up a very small percentage of total proteins in thesample. For purposes of quantitation, the samples should contain similaramounts of proteins. Typically, solid supports with high protein bindingcapacity, e.g., nitrocellulose, are used, and both the primary andsecondary antibodies are used in excess. Those of ordinary skill in theart using routine experimentation will be able to determine the optimalassay conditions required for detection of osteopontin in the samples.

Solid phase supports suitable for use in the assays will be known tothose of ordinary skill in the art. These include microtiter wells,membranes, beads, magnetic beads, discs, gels, flat sheets, test strips,fibers and other configurations and types of materials that permitantigens and antibodies to be attached to the support. Attachment may bemade by noncovalent or covalent means. Preferably, attachment will bemade by absorption of the antibody or antigen to a well in a microtiterplate or to a membrane such as nitrocellulose. These techniques arefamiliar to those skilled in immunology and are well known in the art.

To determine the presence or absence of mesothelioma in a patientaccording to the methods of this disclosure, the level of osteopontin,detected by methods such as those disclosed herein, is typicallycompared to a predetermined value that is capable of distinguishing thepresence of mesothelioma from non-mesothelioma sequelae of asbestosexposure in a specified patient population, for example a populationwith a history of asbestos exposure. The predetermined value may be anempirically determined value or range of values determined from testmeasurements on groups of patients with a particular class of tumor,e.g., other lung tumor. The predetermined value may allow identificationof patients with any of Stage I, Stage II or Stage III mesothelioma. Apredetermined value may also be of use in screening for patients havingStage I mesothelioma, and more particularly prior to appearance ofsymptoms in the patient. Alternatively, the predetermined value may bebased on values measured in a particular patient over a period of time.The Examples below illustrate methods by which a predetermined value forserum osteopontin levels may be determined in patients with and withoutmesothelioma. It should be understood by those of ordinary skill in theart that such methods are routine and can be used without undueexperimentation with other classes of tumors and with fluids other thanserum, and are expected to be useful in human and non-human mammals.

In another suitable embodiment, the predetermined value is determinedusing a Receiver Operator Curve. This method may be used to arrive atthe most accurate cut-off value, taking into account the false positiverate and the false negative rate of the diagnostic assay.

The assay can be performed in a flow-through or strip-test format byimmobilizing the binding agent in a membrane. In a flow-through test,the sample is passed through the membrane and osteopontin contained inthe sample complexes with the binding agent. A solution containing asecond labeled binding agent is passed through the membrane and theamount of the detection reagent that binds to the complex is determined.In the strip test method, the membrane containing immobilized bindingagent is dipped into a fluid sample from the patient. The samplemigrates along the membrane through a region containing a second bindingagent to the area containing immobilized binding agent. The amount ofimmobilized binding agent is selected to generate a visually detectablepattern when the sample contains a specified level of osteopontin.Antibodies and antigen-binding fragments are preferred for use in suchassays, preferably in amounts ranging from 25 ng to about 1 ug, morepreferably from about 50 ng to about 500 ng. Only very small amounts ofpatient samples are required for such a test. Specific examples ofuseful methods can be found in U.S. Pat. Nos. 5,518,869 and 5,712,172.

Nucleic acids including naturally occurring nucleic acids,oligonucleotides, antisense oligonucleotides, and syntheticoligonucleotides that hybridize to the nucleic acid encodingosteopontin, are also useful as agents to detect the presence ofosteopontin in the biological samples (e.g., body fluids) ofmesothelioma patients. The sequence of the human osteopontin gene hasbeen disclosed by others. The present invention includes the use ofnucleic acid sequences corresponding to the coding sequence ofosteopontin and to the complementary sequence thereof, as well assequences complementary to the osteopontin transcript sequencesoccurring further upstream or downstream from the coding sequence (e.g.,sequences contained in, or extending into, the 5′ and 3′ untranslatedregions) for use as agents for detecting the expression of osteopontinin biological samples obtained from human subjects, such as humansputum, bronchial lavage fluid, blood, or serum obtained from a patientat risk for development mesothelioma.

The preferred oligonucleotides for detecting the presence of osteopontinare those that are complementary to at least part of the cDNA sequenceencoding osteopontin. These complementary sequences are also known inthe art as “antisense” sequences. These oligonucleotides may beoligoribonucleotides or oligodeoxyribonucleotides. In addition,oligonucleotides may be natural oligomers composed of the biologicallysignificant nucleotides, i.e., A (adenine), dA (deoxyadenine), G(guanine), dG (deoxyguanine), C (cytosine), dC (deoxycytosine), T(thymine) and U (uracil), or modified oligonucleotide species,substituting, for example, a methyl group or a sulfur atom for aphosphate oxygen in the inter-nucleotide phosphodiester linkage.Additionally, these nucleotides themselves, and/or the ribose moietiesmay be modified.

The oligonucleotides may be synthesized chemically, using any of theknown chemical oligonucleotide synthesis methods well described in theart. For example, the oligonucleotides are prepared by using any of thecommercially available, automated nucleic acid synthesizers.Alternatively, the oligonucleotides may be created by standardrecombinant DNA techniques, for example, inducing transcription of thenoncoding strand. The DNA sequence encoding osteopontin may be invertedin a recombinant DNA system, e.g., inserted in reverse orientationdownstream of a suitable promoter, such that the noncoding strand now istranscribed.

Although any length oligonucleotide may be utilized to hybridize to anucleic acid encoding osteopontin, oligonucleotides typically within therange of 8-100 nucleotides are preferred. Most preferableoligonucleotides for use in detecting osteopontin in urine samples arethose within the range of 15-50 nucleotides.

The oligonucleotide selected for hybridizing to the osteopontin nucleicacid, whether synthesized chemically or by recombinant DNA technology,is then isolated and purified using standard techniques and thenpreferably labeled (e.g., with ³⁵S or ³²P) using standard labelingprotocols.

The present invention also includes the use of oligonucleotide pairs inpolymerase chain reactions (PCR) that are qualitative, semiquantitative,or nonquantitative to detect the expression of osteopontin in biologicalfluids. The oligonucleotide pairs consist of a osteopontin primer and areverse osteopontin primer.

The presence of osteopontin in a sample of biological fluid of a patientcan be determined by nucleic acid hybridization, such as but not limitedto Northern blot analysis, dot blotting, microarray hybridization,Southern blot analysis, fluorescence in situ hybridization (FISH), andPCR. Chromatography, preferably HPLC, and other known assays may also beused to determine messenger RNA levels of osteopontin in a sample.

The osteopontin DNA conceivably may be found in the biological fluidsinside a osteopontin-positive cancer cell that is being shed or releasedin the fluid under investigation.

In one aspect, the present invention contemplates the use of nucleicacids as agents for detecting osteopontin in biological fluids ofpatients, wherein the nucleic acids are labeled. The nucleic agents maybe labeled with a radioactive label, a fluorescent label, an enzyme, achemiluminescent tag, a calorimetric tag or other labels or tags thatare discussed above or that are known in the art.

In another aspect, the present invention contemplates the use ofNorthern blot analysis to detect the presence of osteopontin mRNA in asample of bodily fluid. The first step of the analysis involvesseparating a sample containing osteopontin nucleic acid by gelelectrophoresis. The dispersed nucleic acids are then transferred to anitrocellulose filter or another filter. Subsequently, the labeledoligonucleotide is exposed to the filter under suitable hybridizingconditions, e.g. 50% formamide, 5×SSPE, 2×Denhardt's solution, 0.1% SDSat 42EC., as described in Molecular Cloning: A Laboratory Manual,Maniatis et al. (1982, CSH Laboratory). Other useful procedures known inthe art include solution hybridization, dot and slot RNA hybridization,and probe based microarrays. Measuring the radioactivity of hybridizedfragments, using standard procedures known in the art quantitates theamount of osteopontin nucleic acid present in the biological fluid of apatient.

Dot blotting involves applying samples containing the nucleic acid ofinterest to a membrane. The nucleic acid can be denatured before orafter application to the membrane. The membrane is incubated with alabeled probe. Dot blot procedures are well known to the skilled artisanand are described more fully in U.S. Pat. Nos. 4,582,789 and 4,617,261,the disclosures of which are incorporated herein by reference.

Polymerase chain reaction (PCR) is a process for amplifying one or morespecific nucleic acid sequences present in a nucleic acid sample usingprimers and agents for polymerization and then detecting the amplifiedsequence. The extension product of one primer when hybridized to theother becomes a template for the production of the desired specificnucleic acid sequence, and vice versa, and the process is repeated asoften as is necessary to produce the desired amount of the sequence.

A specific example of PCR that is routinely performed by the skilledartisan to detect desired sequences is reverse transcript PCR. RT-PCRinvolves isolating total RNA from biological fluid, denaturing the RNAin the presence of primers that recognize the desired nucleic acidsequence, using the primers to generate a cDNA copy of the RNA byreverse transcription, amplifying the cDNA by PCR using specificprimers, and detecting the amplified cDNA by electrophoresis or othermethods known to the skilled artisan. Other suitable methods ofdetecting osteopontin nucleic acid in biological samples includeNorthern blot analysis, dot blotting, Southern blot analysis, FISH, andPCR.

The above descriptions are exemplary only, and are not intended to limitthe scope of the disclosure in any way. It is recognized that thoseskilled in the art will know of other types of assays that are suitablefor use in measuring osteopontin, its fragments and variants.

The methods of the present disclosure are also useful for monitoring theresponse of a patient's mesothelioma to therapy, and for predicting therisk of relapse following therapy. With the use of the noninvasiveinventive methods described herein, osteopontin levels can be followedfrom the time that mesothelioma is first diagnosed in a patient throughvarious stages of therapy and following therapy to assess the likelihoodof relapse. Furthermore, the pretreatment levels of osteopontin inpatients with mesothelioma are useful prognostic indicators in thesepatients.

The present disclosure also encompasses the use of osteopontinmeasurements in combination with measurements of other selectedcancer-related proteins such as, for example, including but not limitedto PAI-1, uPA, uPAR, TF, VEGF, adrenomedullin, transforming growthfactor-alpha, soluble mesothelin related peptides (SMRP), CA-125, CYFRA,and other gene products, for the diagnosis, prognosis, and therapy ofcancer already discovered or in discovery phase. In this regard,microarray technology is a convenient approach, although otherapproaches can be used.

The diagnostic and prognostic methods herein may be used alone or incombination with other methods of detecting asbestos-related neoplasms.In some embodiments, compounds used to detect osteopontin can becombined with compounds used to detect other asbestos-related neoplasms,for example in a kit form, either together in one container or detectionsurface, or provided separately. For example, U.S. Pat. No. 4,569,788discloses monoclonal antibodies for detecting non-small cell lungcancer, and U.S. Pat. No. 6,902,890 discloses methods and compositionsfor detecting lung cancers. Such methods and compositions can also beused in association with the present methods in order to determine if asubject has mesothelioma or another neoplasm of the lung. These methodsand compositions are also of use in monitoring a patient's response tochemotherapy, and any other treatment of asbestos-related neoplasmsincluding mesothelioma.

The disclosure also encompasses a method of assessing mesothelioma in ahuman patient, the method comprising assessing osteopontin in a pleuralfluid, whereby an abnormally high level of osteopontin in the pleuralfluid is an indication that the patient is afflicted with mesothelioma.The pleural fluid can be selected from the group consisting of pleuralexudates, pleural transudates, pleural washes, pleural aspirates, andcombinations thereof. The pleural fluid may be collected from a patientafflicted with pleural effusion, or from a patient suspected ofexhibiting pleural effusion. The pleural fluid can be collected from thepatient by thoracentesis, for example using methods know in the art.

In another aspect, the present disclosure encompasses screens formesothelioma-related therapies based on measurement of osteopontinlevels in bodily fluids of animal tumor models of mesothelioma, as knownin the art.

The present disclosure includes a kit for use in carrying out themethods of this disclosure comprising at least one binding agent (andoptionally a detecting agent) for detecting a level of osteopontin in afluid sample from a patient with mesothelioma and a calibration meansfor comparing the level with a predetermined value or values.

The aspects of the disclosure described herein are intended for use inhuman and veterinary medicine.

The following examples are presented solely for illustration, and not tolimit the scope of the claims.

EXAMPLES Example 1 Patient Population Asbestos-Exposed Group

Serum was obtained after written informed consent from 69 persons with ahistory of asbestos exposure and/or radiographic changes consistent withasbestosis at the Center for Occupational and Environmental Medicine,Royal Oak, Mich. from July 2004-September 2004. Entry criteria for thiscohort were similar to that described by Cullen (Cullen M R, Am JEpidemiol 2005; 161(3):260-270). Asbestos exposure and its duration weredocumented using the ATS —Division of Lung Diseases (DLD)-78 Adultquestionnaire (Ferris B G. Am Rev Respir Dis 1978; 118(6 Pt 2):1-120).

Subjects were either employed in a trade with established habitualasbestos exposure for which there is a documented increased risk ofasbestos related diseases (including insulation, sheet metal work,plumbing, plasterboard application, ship fitting, ship electrical work,boiler making, or ship scaling) (Selikoff I J, et al. CA Cancer J Clin1984; 34(1):48-56) or had occupational asbestos exposure (determined byATS-Division of Lung Diseases (DLD)-78 Adult questionnaire) in any jobor occupation and evidence of radiographic changes consistent with adiagnosis of nonmalignant asbestos-related disease. These radiographicfindings included: benign pleural disease, defined as thickening orfibrotic plaques on pleural surfaces of the lung bilaterally, and/ordiffuse lung scarring manifested by small irregular shadows bilaterally.

Each subject had a plain chest radiograph which was interpreted by asingle trained radiologist (National Institute for Occupational Safetyand Health, “B-reader”) with proficiency in the classification of chestradiographs for pneumoconiosis using the International Labor Office(ILO) Classification System. The B reader specifically commented on thepresence or absence of pleural changes, including plaques, and lungfibrosis. Lung fibrosis was interpreted using the InternationalClassification of Radiographs of Pneumoconiosis (available at the ILOweb site). This 12 point system classifies fibrosis by size and numberof abnormal areas (0/− to 3/+) and only readings of 1/0 or greater wereclassified as asbestosis.

To document serum osteopontin levels in an unexposed, but similarpopulation, we obtained serum after informed consent from 45 current(25) or former smokers undergoing screening bronchoscopy as entrycriteria for a chemoprevention trial. Occupational histories wererecorded on all these subjects (age 33-74 years) to document the absenceof known exposure to asbestos; all these participants had normal a chestradiograph.

Of the 69 subjects in the asbestos-exposed group, 57 (83%) had anexposure in an asbestos related trade for 5 years or more, 7 (10%) hadsuch exposure for less than five years, and 5 (7%) had radiographicabnormalities consistent with asbestos exposure but none documented byinterview. The professions of the 64 participants with asbestos exposurewere: foundry-iron workers (11), pipe fitters (7), building andconstruction (7), passive exposure in construction or from family (6),brake assembly/repair (5), boiler repair (4), vermiculite insulationexposure (4), machinist grinder (3), plumber (3), tool and die industry(2), ship builder (2), millwright (2), firefighter (2), brick maker (2),electrician (2), and asbestos remover (2). Radiographic evidence offibrosis was seen in 23/69 (33%), and pleural plaques were found in50/69 (73%) subjects; six participants with 5 to 37 years of exposurehad no radiographic abnormalities, 53 had either plaque or fibrosis, and10 had both plaques and fibrosis.

Pleural Mesothelioma

Serum was obtained after written informed consent from 76 patients underan approved Wayne State University Human Investigation Protocol (D1420)before cytoreductive surgery for pleural mesothelioma. The oldest serumwas obtained 77 months before analysis, while the most recent was 3months old. Asbestos exposure was documented by occupational history in59/76 (78%) of the mesothelioma patients. All patients had completesurgical staging according to the International Mesothelioma InterestGroup (IMIG) staging system (Rusch V W. Lung Cancer 1996; 14(1):1-12)(Stages I-13, 11-20; III-43), and were followed with computerizedtomography of the chest every three to four months until death. Tumorswere classified as epithelial (50), sarcomatoid (4), or mixed (22).Table 1 lists characteristics of the asbestos exposed and mesotheliomagroups.

TABLE 1 Demographics of Mesothelioma and Asbestos Exposed PopulationsMalignant Pleural Asbestos-Related Mesothelioma (76) NonmalignantDisease (69) Age, years 65 + 1 65 + 1 (mean + standard error) Sex 60Male/16 Female 61 Male/8 Female Ethnicity 72 Caucasian/4 African- 66Caucasian/3 African- American American Smoking History 60 Smoker/16 56Smoker/13 Non-smoker Non-smoker

Statistical Analyses

Kaplan Meier survival plots and log rank tests were used to assessdifferences in survival for the pleural mesothelioma patients. Theability of serum osteopontin levels to distinguish the pleuralmesothelioma group from the asbestos-related group was evaluated bydescriptive statistics, and by Receiver Operating Characteristic (ROC)curves (Metz C E. Semin Nucl Med 1978; 8(4):283-298; Zweig M H, et al.Clin Chem 1993; 39(4):561-577). The area under the ROC curve (AUC) wascalculated, and 95% confidence intervals used to test the hypothesisthat the theoretical area is 0.5. An AUC whose confidence interval didnot include the 0.5 value was considered evidence that the laboratorytest had some ability to distinguish between the mesothelioma andasbestosis-exposed groups (Zweig M H, et al. Clin Chem 1993;39(4):561-577; Hanley J A, et al. Radiology 1982; 143(1):29-36).Differences between groups were calculated using ANOVA and by multipleregression analysis in stepwise fashions entering only variables thatwere p<0.05 in the model. All statistical analyses were performed usingMedCalc Software, Mariakerke, Belgium.

Example 2 Survival by Stage of Pleural Mesothelioma

To determine whether the mesothelioma patients in this study hadoutcomes similar to those in other series, survival and time toprogression curves based on IMIG staging status were generated. FIG. 1shows significant differences in survival and progression according tostage as follows: (A) median survivals for the 13 Stage I, 20 Stage II,and 40 Stage III patients were 32, 19, and 13 months, respectively,p=0.006 by log rank test; (B) median time to progression for the 13Stage I, 20 Stage II, and 40 Stage III patients were 23, 14, and 7months, respectively, p<0.001 by log rank test. These results areconsistent with those of other studies that used the IMIG staging system(Pass H I, et al. J Thorac Cardiovasc Surg 1998; 115(2):310-317; Rusch VW, et al. Ann Thorac Surg 1999; 68(5):1799-1804; Rusch V W, et al. JThorac Cardiovasc Surg 1996; 111(4):815-825).

Example 3 Immunohistochemistry

Immunohistochemistry was performed on a multi-tissue pleuralmesothelioma array, consisting of 2-mm representative areas of resectedtumor and normal tissue controls. Thirty-eight of the 76 pleural tumorsstudied for serum osteopontin were spotted on the array. The other 38were not available at the time of array construction.Immunohistochemistry was performed using the standard ABC technique. Theprimary antibody, a monoclonal anti-osteopontin antibody (clone OP3N,Vector Laboratories, Burlingame, Calif.), was applied to the array at1:150 dilution, for 90 minutes at room temperature. The secondary antimouse IgG (Vector Laboratories, Burlingame, Calif.) was applied at 1:200dilution for 30 minutes at room temperature. A positive control, and anegative control (obtained by omitting the secondary antibody) wereincluded in each run. Cases were scored separately for (1) intensity (I)(scale of 1 to 3) and (2) extent (E) of positive tumor cells (1≦10%;2=≧10 and ≦50%; 3=≧50%).

Of the tumor tissue available for osteopontin staining (from 38 of the76 mesotheliomas), 36 of the 38 were positive for osteopontin (FIG. 2).They showed cytoplasmic staining in ≧50% of tumor cells, and stainingintensity ranged from 1 to 3 (13=1; 8=2; 15=3). Osteopontin was seen inall pleural mesothelioma variants (epithelial, 19/20; biphasic 15/16;and sarcomatoid 2/2). Lung parenchyma and adjacent normal pleura werenegative; fibroblasts in tumor-associated stroma were infrequentlyweakly positive; the media and intima of vessels showed weak positivity.In FIG. 2A, magnification was ×100; in FIG. 2B, magnification was X 400.

Example 4 ELISA Assay for Osteopontin

For osteopontin ELISA, the Human Osteopontin Assay Kit (IBL Company,LTD., Gunma, Japan) was used to determine the level of serumosteopontin; all samples were coded. Each specimen was tested induplicate and the results quantitated in ng/ml using a standard curve.

Asbestos-Exposed and Unexposed Groups

The mean serum level of osteopontin in the entire asbestos-exposed groupwas 30±3 ng/ml (range 2-221 ng/ml, 95% Confidence Interval (CI)=23.1 to36.1 ng/ml). The levels in age-matched unexposed controls with normalradiographs did not differ from those measured for the asbestos exposedgroup (age <50: 12±5 ng/ml vs. 25±11 ng/ml, respectively, 95% CI ofdifference −16 to 41 ng/ml, p=0.34; age 50-60: 19±6 ng/ml vs 24±5 ng/ml,respectively, 95% CI of difference −12 to 22 ng/ml, p=0.56; age >60,24±5 ng/ml vs 32±4 ng/ml, respectively, 95% CI of difference −23 to 7,p=0.29 (FIG. 3A). For the asbestos-exposed cohort, there were nosignificant differences in osteopontin levels according to sex or thepresence or absence of pleural plaques (p=0.19, and p=0.88,respectively; FIGS. 3B, D).

The subgroup with lung fibrosis had a significantly higher mean level ofosteopontin than the subgroup without fibrosis (43 ng/ml vs. 23 ng/ml,respectively, 95% CI of difference 7 to 33 ng/ml. p=0.004), and the meanlevels were significantly higher with 10 or more years of exposure (34ng/ml vs. 16 ng/ml, respectively, 95% CI of difference 4 to 33 ng/ml,p=0.015, FIG. 3C). The highest levels of serum osteopontin were found insubjects who had both plaques and fibrosis (56±13 ng/ml).

When serum osteopontin levels for age matched unexposed controls (age,64±3 years) were compared to asbestos exposed subjects with plaques andfibrosis (age, 64±3 years), significant differences in osteopontinlevels were noted (14±6 ng/ml vs. 56±13 ng/ml, p=0.03). Asbestos exposedsubjects with a normal chest radiograph (21±5 ng/ml), plaques (23±3ng/ml), or fibrosis (32±7 ng/ml) also had significantly lowerosteopontin levels than those with plaques and fibrosis (p=0.004). Amultiple regression analysis that included age, duration of asbestosexposure, presence of fibrosis, presence of plaques, and theInternational Labor Organization radiography score was performed. Onlythe duration of asbestos exposure (p=0.001) and the radiography score(p<0.001) were independently associated with osteopontin levels, withzero order correlation coefficients of 0.357 and 0.399 respectively.

Pleural Mesothelioma

The mean serum osteopontin level in the mesothelioma group, 133±10 ng/ml(range 6-385 ng/ml, 95% confidence interval 113-154 ng/ml), wassignificantly different from the mean in the asbestos-exposed group(30±3 ng/ml, range 2-221 ng/ml, 95% CI=23.1 to 36.1 ng/ml, p<0.001).There were no significant differences in mean serum osteopontin levelsin Stage I (147±26 ng/ml, range 10-341 ng/ml, 95% CI 92-204 ng/ml),stage II (158±22 ng/ml, range 14-385 ng/ml, 95% CI 99-216 ng/ml)), orstage III (118±12 ng/ml, range 4-302 ng/ml, 95% CI 83-145 ng/ml)mesothelioma (P=0.15, Stage II vs. Stage III,); however, the means inall these stages differed significantly from the mean of theasbestos-exposed group (30±3 ng/ml, range 2-221 ng/ml, 95% CI=23.1 to36.1 ng/ml, p<0.001). Moreover, when serum osteopontin levels for theasbestos exposed subjects with plaques and fibrosis were compared withlevels from the pleural mesothelioma group, the levels weresignificantly different (56±13 ng/ml vs 133±10 ng/ml, respectively, 95%CI of difference=49 to 114 ng/ml, p<0.0001). Mean osteopontin levelswere similar in men and women with mesothelioma (136±12 ng/ml vs. 125±21ng/ml, respectively; 95% CI of difference=−61 to 39 ng/ml, p=0.6575) anddid not vary by tumor histology (epithelial, 128±13 ng/ml vs.non-epithelial, 133±18, 95% CI of difference=−28 to 59 ng/ml, p=0.49) orhistory of asbestos exposure (exposed 151±24 ng/ml vs. unexposed 128±12ng/ml, 95% CI of difference=−73 to 28 ng/ml, p=0.37).

Example 5 Receiver Operating Characteristic Curves

ROC analyses comparing the asbestos exposed subjects to the pleuralmesothelioma patients had an area under the curve (AUC) of 0.888 (95% CI0.825 to 0.934) (FIG. 4).

The six panels of FIG. 4 are as follows: (A) All 76 mesotheliomas versus69 asbestos exposed, Area Under Curve (AUC)=0.888. (B) High sensitivityfor early detection of mesothelioma at a cutoff level of 10.9 ng/ml. (C)Cut-off for minimization of total classification error was level >48.4ng/ml. (D) Stage I mesotheliomas versus asbestos exposed subjects,AUC=0.906. (E) High sensitivity for early detection of Stage Imesothelioma versus asbestos exposed subjects at cutoff level of 9.5ng/ml. (F) Cut-off for minimization of total classification error forStage I versus asbestos exposed was a level >62.4 ng/ml.

Subgroup analyses revealed AUCs for Stage I, Stage II, Stage I/II, andStage III pleural mesothelioma, compared with asbestos controls, to be0.906, 0.925, 0.917, and 0.865, respectively. A cut-off of 48.4 ng/ml(sensitivity and specificity of 78% and 87%) had the highest accuracy(minimal false negative and false positive) for confirming mesotheliomafrom the asbestos exposed cohort without mesothelioma. For the purposeof screening, i.e. early detection for mesothelioma, a cutoff with thehighest (95-99 percent) sensitivity might be most appropriateindependent of specificity, and at a cutoff of 10.9 ng/ml, thesensitivity for osteopontin level was 96.1% with a specificity of 23.2%.If screening were for detection of Stage I disease only, a cutoff of 9.5ng/ml gave 100% sensitivity with 20.3% specificity. The most accuratecutoff, however, for the detection of a Stage I mesothelioma(sensitivity of 84.6% and specificity of 88.4%) was with a cut-off of62.4 ng/ml.

All references cited herein are fully incorporated by reference. Havingnow fully described the disclosure, it will be understood by those ofskill in the art that the disclosure may be performed within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the disclosure or any embodimentthereof.

1. A method of diagnostically evaluating a human subject for thepresence of pleural mesothelioma, comprising: (a) obtaining a biologicalsample from said subject; (b) assaying said sample for the concentrationof osteopontin present; (c) comparing the results obtained from theassay of step (b) with results obtained from the assay of one or morecontrol samples; and (d) concluding that said subject is at increasedrisk of having pleural mesothelioma if the concentration of osteopontinin said subject sample is higher than the concentration in said controlsample or samples.
 2. The method of claim 1, wherein the concentrationof osteopontin present in said samples is determined by an immunoassay.3. The method of claim 1, wherein the concentration of osteopontin insaid sample is determined using an ELISA.
 4. The method of claim 1,wherein said biological sample is serum.
 5. The method of claim 1,wherein said one or more control samples are obtained fromasbestos-exposed subjects.
 6. The method of claim 1, wherein the resultsfrom said subject sample is compared to the results obtained from agroup of control samples taken from the general population.
 7. Themethod of claim 1, wherein it is concluded that a subject is atincreased risk of having mesothelioma if the osteopontin concentrationin said subject sample is higher than the amount in said control sampleor samples by a predetermined value.
 8. The method of claim 1, furthercomprising performing at least one additional assay for a diagnosticmarker of cancer.
 9. The method of claim 8, wherein said cancer isnon-mesothelioma asbestos-related lung cancer. 10-11. (canceled)
 12. Amethod of diagnostically evaluating mesothelioma treatment in a humansubject following diagnosis of mesothelioma, comprising: (a) obtainingat least a first and a second biological sample from said subject atdifferent time points during the treatment; (b) assaying said samplesfor the concentration of osteopontin present; (c) comparing the resultsobtained from the assay of step (b) with results obtained from the assayof a pretreatment sample from said patient; and (d) concluding that saidtreatment is effective in reducing the severity of mesothelioma if theconcentration of osteopontin in said pretreatment sample is higher thanthe concentration in said first and/or second sample.
 13. The method ofclaim 12, wherein said biological samples are serum samples.
 14. Themethod of claim 13, wherein the pre-treatment serum osteopontin level isat least 60 ng/ml, and the serum osteopontin level of at least one ofsaid first and second samples is between 30 and 58 ng/ml. 15-17.(canceled)
 18. A method of assessing the likelihood that a human patientwill develop mesothelioma, the method comprising assessing the amount ofosteopontin in a sample obtained from the patient and comparing thatamount with a reference value derived from one or more reference humansof known mesothelioma outcome, whereby the comparison indicates thelikelihood that the patient will develop mesothelioma.
 19. The method ofclaim 18, wherein the mesothelioma is pleural mesothelioma.
 20. Themethod of claim 18, wherein the sample comprises serum. 21-44.(canceled)